| As one of the five new traditional Chinese drugs developed Jiangsu Kang Yuan Pharmaceutical Co.,Ltd.,ginkgo diterpene lactone meglumine injection is prepared with ginkgo diterpene lactone as raw materials and meglumine as solubilizing agents.Raw ginkgo diterpene lactone materials are the processed extracts of the dried leaves of ginkgoaceae plant-Ginkgo biloba L,and contain ginkgolide A(GA),ginkgolide B(GB),ginkgolide K(GK)are mainly ingredients.Because of meglumine,ginkgolides mainly exist in the form of hydrolysates in this injection.After administration of the pharmaceutical injection,both hydrolysates and the prototype form of GA,GB,and GK can be found in biological fluids because there is a free conversion between the hydrolysate and prototype in physiological conditions.Moreover,the standard refence metrials of hydrolysed ginkgilides could not be prepared.Pharmacokinetic research of ginkgo diterpene lactone meglumine injection was difficult to carry out.In this work,liquid chromatography-tandem mass spectrometry was used to determine the total amount and the amount of the prototype form of the lactone for GA,GB,and GK by liquid-liquid extraction and acidification with liquid-liquid extraction.The concentrations of the hydrolysed carboxylic forms of ginkgolides were calculated,and the pharmacokinetics of the three forms of GA,GB,and GK were assessed after intravenous administration of GDLI in rats.The distribution,metabolism and excretion process and characteristics of ginkgo diterpene lactone meglumine injection in vivo was systematically stated to reveal the dynamic variation rule of this drug in vivo.The main contents of this research could be summarized as follows:1.The establishment of LC-MS/MS analysis methods for ginkgolide A,B and K in different biological matrixBy the application of LC-MS/MS technology to establish a series of acidified biological sample determination methods for GA,GB and GK in the plasma,bile,urine and feces of rats and the plasma of beagle dogs,the results showed that the specificity,sensitivity,precision,accuracy,extraction recovery rate,matrix effect and sample stability of the above methods were in line with the determination requirements of biological samples.At the same time,acidified plasma accompanying standard curve was used to verify the precision of the un-acidified plasma samples and the result was also consistent with determination requirements.The above description indicated that LC-MS/MS determination methods for ginkgo lactone A,B and K in acidizing plasma could also be used for the determination of un-acidified plasma and-acidified plasma samples.2.Pharmacokinetic research of ginkgo diterpene lactone meglumine injection in ratsIn this part,the time course,tissue distribution,metabolism and excretion dynamic process of ginkgo di-terpene lactone meglumine injection in the plasma of rats were systematically researched and the results were as follows:(1)The plasma time course of single dosing administration:the plasma drug concentration peak(Csmin)of prototype form,hydrolysates and the total amount of GA,GB,GK in rats after single dosing administration was in direct proportion to the systemic exposure and administration dosage,which suggested that the time course behavior of GA,GB and GK in rats after single dosing administration presented linear dynamic process(r>0.9).The concentrations of hydrolysis and prototype form of GA and GB in rats was showed dynamic change in vivo and their proportion gradually decreased with the gradual time extension and reached steady state in the end.(2)The plasma time course of multiple dosing administration:after continuously multiple administration of 3 mg/kg ginkgo diterpene meglumine injection,the elimination half-life of all forms of GA,GB and GK showed no significant difference compared with single dosing administration,which suggested that the absorption rate and elimination rate of the ingredients of ginkgo diterpene lactone meglumine injection in rats had no significant change after multiple dosing administration and explained that it had no accumulation in the bodies of rats(p<0.05).(3)Tissue distribution research:after single intravenous administration of 3 mg/kg ginkgo diterpene lactone meglumine injection(5 min),GA,GB and GK in rats presented highest drug concentration in most tissues and were distributed in most tissues,of which the distribution concentration in the kidney was the highest followed by the liver,intestine,heart,lung,spleen,pancreas,fat,muscle,skin and ovary,et al.The hydrolysates of ginkogildes were exsited in brain,it suggested that the hydrolysates of ginkogildes could penetrate across the blood-brain barrier.(4)Excretion research:after single dosing administration,the total cumulative excretion rates of GA,GB and GK in rats respectively were approximately 50.3%,70.4%and 86.1%,of which GB presented the relatively high cumulative excretion score-about 43.9%in the urine,which indicated that it might be mainly excreted through kidney;GK presented the higher cumulative excretion score-about 63.7%in the urine,which indicated that it might be mainly excreted through intestine.The excretion of total amounts of GB and GK approached to mass balance.3.Pharmacokinetic research of ginkgo diterpene lactone meglumine injection in beagle dogsIn this part,the time course of ginkgo diterpene lactone meglumine injection in the plasma of beagle dogs after single and multiple dosing administration was systematically researched and the results were as follows:(1)The plasma time course of single dosing administration:after single dosing administration,the plasma drug concentration peak(C3min)of prototype form,hydrolysates and the total amount of GA,GB and GK was in direct proportion to the systemic exposure and administration dosage(r>0.9),which suggested that the time course behavior of GA,GB and GK in beagle dogs after single dosing administration presented linear dynamic process.The hydrolysis and prototype form of GA and GB in beagle dogs showed dynamic change in vivo and the proportion of concentrations gradually reduced with the gradual time extension and reached steady state in the end.(2)The plasma time course of multiple dosing administration:after continuous administration of lmg/kg ginkgo diterpene meglumine injection for 7 days,the elimination half-life of all forms of GA,GB and GK in beagle dogs showed no significant difference compared with single dosing administration,which suggested that the absorption rate and elimination rate of the ingredients of ginkgo diterpene lactone meglumine injection in beagle dogs had no significant change after multiple dosing administration and explained that it had no accumulation in the bodies of beagle dogs(p>0.05).4.The study on metabolism and drug-drug interaction of ginkgo diterpene meglumine injectionIn this part,the protein binding rate,in vitro metabolism as well as transporters and liver enzyme based drug interactions of the main effective components of Ginkgo diterpene lactone meglumine injection,GA,GB and GK,were systematically studied.The results were as follows:(1)The plasma protein binding rates of GA,GB and GK:within the selected dose range,the separate administration and mixed administration of the plasma protein binding rates of GA,GB and GK had no significant difference,indicating that there was no competitive inhibition between them.The proteins binding rate of GA,GB and GK were between 26.5 to 53.0%in rat plasma,between 21.4 to 46.4%in Beagle dog plasma and between 21.0 to 60.3%in human plasma,suggesting that drug interactions might not be induced.(2)The cellular uptake characteristics of GA and GB:the Caco-2 and HepG2 cell models were used to observe the influence of drug concentration,incubation time and temperature on the uptake of GA and GB.The results showed that within the selected concentration range,the uptake of GA and GB showed a concentration dependence and could quickly reach the balance.The uptake of GB was not affected by temperature while GA was greatly affected by temperature.(3)The effect of GA and GB on the p-glycoprotein:the HepG-2,Caco-2 and MCF-7/A cell models were used to evaluate the interaction between GA,GB and P-gp.The results showed that GA and GB were not the inhibitors of P-gp and GB was also not a specific substrate for P-gp,but GA might be a substrate for P-gp,.(4)In vitro metabolic stability of GA,GB and GK:the stability of GA,GB and GK in different kinds of liver microsomes were studied and their metabolites were analyzed.The results showed that GA and GB had a relative stability in liver microsomes while GK had a large different stability.And two metabolites of GK were found in all kinds of liver microsomes.(5)Evaluation of the inhibitory effects of GA,GB and GK on the human P450 enzyme:the liver microsomal incubation technique was used to evaluate the inhibitory effects of GA,GB and GK on the subtype of P450 enzyme.The results showed that GA,GB and GK had no significant inhibitory effect on the 6 kinds of metabolic enzymes,indicating the lower probability of drug interactions. |
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